Method of viral inhibition

ABSTRACT

This invention relates to methods of treating or preventing viral infections caused by flaviviruses, such as dengue virus, yellow fever virus, West Nile virus or Japanese encephalitis virus or infections caused by Chikungunya virus (CHIKV). The methods involve the administration of retinoic acid analogues to subjects who have, are suspected of having a flavivirus infection or infection with CHIKV, or to those who are at risk of becoming infected with a flavivirus or becoming infected with CHIKV.

RELATED APPLICATIONS

This application is a US National stage entry of InternationalApplication No. PCT/AU2014/050017, which designated the United Statesand was filed on Apr. 16, 2014, published in English. This applicationclaims priority under 35 U.S.C. § 119 or 365 to Australian ApplicationNo. 2013901525, filed on Apr. 16, 2013. The entire teachings of theabove applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a method of treating viral infections causedby flaviviruses, such as dengue virus, yellow fever virus, West Nilevirus or Japanese encephalitis virus. In particular, it relates tomethods and compounds for treating dengue virus infection, includingprevention of infection by dengue virus.

BACKGROUND OF THE INVENTION

Arbovirus is a term used to refer to a group of viruses that aretransmitted by arthropod vectors such as mosquitoes and ticks.Flaviviruses are among those transmitted in this way, and include WestNile virus, dengue virus, tick-borne encephalitis virus, and yellowfever virus. Of these, dengue virus is considered one of the mostsignificant arboviruses, with an estimated 390 million cases of denguefever occurring worldwide. The alphavirus Chikungunya virus (CHIKV) isanother example of an arbovirus. Most of these infections are intropical areas of the world.

Like many of the flavivirus infections, symptoms of having been infectedwith dengue virus include sudden high fever, severe headaches, painbehind the eyes, a rash, severe joint and muscle pain, nausea andvomiting and mild bleeding. In more severe cases, infected individualsmay develop dengue hemorrhagic fever, a rare complication characterizedby more severe bleeding, damage to lymph and blood vessels, enlargementof the liver, and failure of the circulatory system, all of which can inturn lead to shock and death.

Currently infections are treated symptomatically as there is no licensedvaccine or effective anti-viral. Moreover dengue fever is caused by oneof five closely related, but antigenically distinct, virus serotypes(DENV-1, DENV-2, DENV-3, DENV-4 and a recently identified single isolateDENV-5), and infection with one serotype may not protect a subject frominfection with a different serotype.

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

SUMMARY OF THE INVENTION

Infections from flaviviruses such as dengue virus serotypes 1-4(DENV1-4), West Nile virus (WNV) and Japanese encephalitis virus (JEV)account for as many as 200,000 deaths/year worldwide. Although DENVinfections have increased globally in the last 50 years, with now >40%of the world's population estimated to be at risk, there is noefficacious vaccine or anti-viral therapeutic currently available tocombat DENV.

CHIKV infection causes an illness with an acute febrile phase lastingtwo to five days, followed by a longer period of joint pains in theextremities. The pain associated with CHIKV infection of the joints maypersist for weeks or months, or in some cases years especially in theelderly. Like many arbovirus infections, there is no specific treatmentaimed at clearing the virus.

Using a high throughput assay to screen for compounds that interferewith specific protein-protein interactions, the inventors haveidentified a retinoic acid analogue and structurally related retinoicacid analogues that have efficacy in the treatment or prevention offlaviviruses, and in particular the treatment or prevention of denguefever, caused by DENV. These “anti-DENV agents” are effective againstall 4 circulating serotypes of DENV (DENV1-4) and can be used eitherprophylactically to protect individuals in DENV-infested regions, or asefficacious therapeutics post-infection.

Accordingly, in a first aspect of the invention, there is provided amethod of treating flavivirus infection, preferably dengue virus (DENV)infection, comprising administering an effective amount of a retinoicacid analogue to a subject in need thereof. Typically the amountadministered achieves a therapeutically effective level in the subject.In an alternative embodiment of this aspect of the invention, theflavivirus infection treated is infection caused by yellow fever virus(YFV), WNV, or JEV.

It is also envisaged that subjects having CHIKV infection will benefitfrom administration of an effective amount of the retinoic acid analogueof the invention.

The words “treat” or “treatment” refer to therapeutic treatment whereinthe object is to slow down (lessen) an undesired physiological change ordisorder. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to alleviation of symptomsof dengue virus infection and stabilised (i.e., not worsening orprogressing) dengue virus infection.

The phrase “effective amount” or “therapeutically effective level” andsimilar terms means an amount of the retinoic acid analogue such asN-(4-hydroxyphenyl) retinamide (4-HPR) that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, (iii) prevents or delays the onset of one or more symptoms ofthe particular disease, condition, or disorder described herein, (iv)prevents or delays progression of the particular disease, condition ordisorder, or (v) reverses damage caused prior to treatment to someextent. The reversal does not have to be absolute. The amountadministered may be higher than what is required within the body toachieve the therapeutic effect, but takes in to account the absorption,distribution, metabolism and excretion (ADME) profile of the analogue,the route of administration and the administration frequency.

In an alternative embodiment, the severity of symptoms associated withflavivirus infection in a subject, preferably DENV infection, can bereduced by administering a therapeutically effective amount of aretinoic acid analogue to the subject. As explained above in the contextof treatment, by “reducing symptom severity” it is meant that one ormore symptoms are attenuated, ameliorated, or eliminated, or the onsetof one or more symptoms is prevented or delayed.

Symptom severity may also be reduced in a subject infected with CHIKV.

In a subject suspected of having flavivirus, or a subject with symptomsof being infected with a flavivirus such as DENV, YFV, WNV, or JEV, adiagnostic or confirmation test may be conducted either prior to orafter the commencement of retinoic acid analogue administration.

In another aspect of the invention there is provided a method ofpreventing flavivirus infection, comprising administering an effectiveamount of a retinoic acid analogue to a subject in need thereof.Typically, the amount administered achieves a prophylactically effectivelevel in the subject, thereby preventing flavivirus infection.

In an alternative embodiment of the invention, there is provided amethod of preventing CHIKV infection, comprising administering aneffective amount of a retinoic acid analogue to a subject in needthereof. Typically, the amount administered achieves a prophylacticallyeffective level in the subject, thereby preventing CHIKV infection.

By “an effective amount” or similar terms, in the context of preventinginfection it is meant the amount of retinoic acid analogue administeredthat is sufficient to achieve a prophylactically effective level whereinthat level protects the subject from becoming infected with aflavivirus, preferably DENV, YFV, WNV or JEV, and most preferably DENV.The amount administered may be higher than what is required within thebody to achieve the preventive/prophylactic effect, but takes in toaccount the ADME profile of the analogue, the route of administrationand the administration frequency.

In preferred embodiments, the flavivirus is DENV, and may be one or moreof serotypes 1, 2, 3 and 4.

In a further embodiment of the invention, there is provided a method forpreventing antibody dependent enhanced (ADE) DENV infection, comprisingadministering an effective amount of a retinoic acid analogue to asubject in need thereof. Typically the amount administered achieves atherapeutically effective level in the subject to reduce the risk of ADEDENV infection. The subject will have had a prior DENV infection with adifferent DENV serotype.

The retinoic acid analogue is preferably N-(4-hydroxyphenyl) retinamide(4-HPR), also known as Fenretinide. 4-HPR may be formulated as acomposition for treatment, or as a composition for prevention. In analternative embodiment, 4-HPR metabolites may be administered. Onepreferred metabolite is N-(4-hydroxyphenyl)-4-oxoretinamide(4-oxo-4-HPR). In yet a further embodiment, the retinoic acid analogueis a 4-HPR variant according to Formula I, as described herein. Each of4-HPR, a 4-HPR metabolite or a 4-HPR variant may be administered aloneas the only active ingredients, or combinations thereof. Whenadministered in combinations, preferably the compounds worksynergistically. All embodiments are contemplated by the invention.

A “therapeutic composition”, “pharmaceutical composition”, “compositionfor treating” or “prophylactic” and other like terms refers to acomposition including a retinoic acid analogue such as 4-HPR, combinedwith appropriate, pharmaceutically acceptable salts, carriers ordiluents.

By “co-administration”, it is meant that the 4-HPR and/or itsmetabolites and/or 4-HPR variants as described herein may be formulatedas a single composition, administered as separate compositionscontemporaneously, or administered consecutively as separatecompositions. Co-administration can also refer to the administration ofthe retinoic acid analogue with a second therapeutic agent, such as painrelievers, to help alleviate the symptoms of infection.

The retinoic acid analogues of the invention may also be prepared as amedicament for the treatment of flavivirus infection, to reduce symptomseverity in a subject having or suspected of having a flavivirusinfection, for the prevention of infection by a flavivirus, or for theprevention of ADE DENV infection.

In an alternative embodiment, the retinoic acid analogues of theinvention may also be prepared as a medicament for the treatment ofCHIKV infection, to reduce symptom severity in a subject having orsuspected of having CHIKV infection or for the prevention of infectionby CHIKV.

There is also provided a retinoic acid analogue or a composition thereofwhen used to treat flavivirus infection, reduce symptom severity in asubject having or suspected of having a flavivirus infection, preventinfection by a flavivirus, or prevent ADE DENV infection.

The invention further provides for a retinoic acid analogue or acomposition thereof for treating flavivirus infection, reducing symptomseverity in a subject having or suspected of having a flavivirusinfection, preventing infection by a flavivirus, or preventing ADE DENVinfection.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. 4-HPR (FIG. 1A) and 4-oxo-4-HPR (FIG. 1B) can specificallyblock association of DENV-2 NS5 with IMPα/β1.

FIG. 2. 4-HPR can specifically block association of DENV-2 NS5 and IMPIMPα/β1, but not DENV-2 NS5 and IMPβ1.

FIGS. 3A-3B. The anti-DENV activity of 4-HPR (FIG. 3A) and 4-oxo-4-HPR(FIG. 3B) is highly specific and not a general activity of retinoic acidanalogues.

FIG. 4. 4-HPR has a highly favourable toxicity profile.

FIGS. 5A-5C. 4-HPR blocks DENV replication when applied to cells priorto (FIG. 5A) and after infection (A, B, C).

FIGS. 6A-6B. 4-HPR specifically blocks replication of DENV serotypes 1-4in the BHK-21 cell line (FIG. 6A) and Huh-7 cell line (FIG. 6B).

FIG. 7. 4-HPR is effective against DENV-1/antibody-dependent enhancedinfection.

FIGS. 8A-8C. 4-HPR is able to protect against DENV infection in PBMCs(FIG. 8A) and in a lethal infection mouse model (FIG. 8B and FIG. 8C).

FIGS. 9A-9C. 4-HPR is effective against the arboviruses CHIKV (FIG. 9Aand FIG. 9B) and WNV (FIG. 9C).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

Dengue virus (DENV) is a mosquito-borne single positive-stranded RNAvirus of the family Flaviviridae, within the genus flavivirus. There are5 antigenically distinct virus serotypes; the four circulating serotypesDENV-1, DENV-2, DENV-3, and DENV-4, and the recently identified singleisolate DENV-5, each of which is able to cause symptomatic infection inhumans. Its genome codes for three structural proteins—capsid protein(C), membrane protein (M) and envelope protein (E)—and sevennonstructural proteins—NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5.

Despite there being a good understanding of the DENV structure andlifecycle, efforts by others in the field prior to the current inventionhave failed to develop an effective vaccine or anti-viral therapy forhumans infected with DENV. Inhibitors targeted exclusively at host cellproteins are problematic in that targeting host proteins often resultsin toxicity, while agents directed at viral components result in viralresistance (Caly et al. 2012). In contrast, targeting host cell:viralprotein interfaces represents a viable strategy to derive antiviralsthat do not kill normal cells or select for virus resistance. Theidentification by the inventors of a retinoid capable of inhibitingdengue virus by targeting host cell:viral protein interactions thereforerepresents a novel and viable treatment for dengue infection. Moreover,the retinoid identified is able to reduce infection when added prior to,as well as post-infection, indicating it could be efficacious bothprophylactically and as a therapeutic.

Treatment and/or prevention (prophylaxis) is aimed at one or more of,without limitation:

-   i) reducing viral load in a subject infected with a flavivirus    including but not limited to DENV, YFV, WNV, or JEV, and in    particular, a person infected with any serotype of DENV.-   ii) reducing or ameliorating the risk to a subject of infection with    a flavivirus including but not limited to DENV, YFV, WNV, or JEV,    and in particular, the risk of infection with any one or more    serotypes of DENV.-   iii) alleviating symptoms of a flavivirus infection in a subject,    having or suspected of having a flavivirus infection including but    not limited to DENV, YFV, WNV, or JEV, and particularly DENV    infection.-   iv) reducing or ameliorating the risk of antibody dependent enhanced    (ADE) DENV infection in a subject having had or suspected of having    had a previous DENV infection.

In alternative embodiments of the invention, each of (i) to (iii) isapplicable to CHIKV.

Accordingly, in one aspect of the invention, there is provided a methodof treating flavivirus infection, preferably DENV, YFV, WNV, or JEVinfection and most preferably DENV, comprising administering aneffective amount of a retinoic acid analogue to a subject in needthereof. Typically the amount administered achieves a therapeuticallyeffective level in the subject.

There is also provided a use of an effective amount of a retinoic acidanalogue in the preparation of a medicament for treating flavivirusinfection, preferably DENV, YFV, WNV, or JEV infection and mostpreferably DENV, in a subject in need thereof. The retinoic acidanalogue is preferably 4-HPR, or one or more 4-HPR metabolites, or 4-HPRand one or more 4-HPR metabolites.

Retinoids are a class of natural and synthetic vitamin A-derivatives inwhich the terminal carboxyl group of retinoic acid is linked to anaminophenol residue. In a particularly preferred embodiment of theinvention, the retinoic acid analogue is N-(4-hydroxyphenyl) retinamide(4-HPR), also known commercially as “Fenretinide”. Fenretinide is knownto be clinically well tolerated and there is substantial interest in itsuse for clinical trials in various cancers, including neuroblastoma andbreast cancer. Due to its low toxicity and effect on various pathwaysincluding ceramide biosynthesis, free radical oxygen and nitric oxidesynthase, 4-HPR has also been investigated as a preventative andtherapeutic agent in various diseases such as diabetes, HIV infection,Alzheimers Disease, cystic fibrosis, allergic encephalomyelitis,ichthyosis and ophthalmic conditions. It has not however been known tohave any therapeutic effects on flaviviruses such as DENV.

In a preferred embodiment of the invention, there is provided a methodof treating flavivirus infection, preferably DENV, YFV, WNV, or JEVinfection and most preferably DENV, comprising administering aneffective amount of a retinoic acid analogue to a subject in needthereof, wherein the amount administered achieves a therapeuticallyeffective level in the subject, wherein the retinoic acid analogueadministered is N-(4-hydroxyphenyl) retinamide.

4-HPR may be administered as a single active, formulated as acomposition for treatment, or as a composition for prevention. 4-HPR maybe metabolised once administered to the subject to producetherapeutically effective metabolites. The metabolites may themselvesalso be administered and be effective at treating or preventingflavivirus infections, such as DENV infection. For example,N-(4-hydroxyphenyl)-4-oxoretinamide (4-oxo-4-HPR). Accordingly, inanother embodiment of the invention, there is provided a method oftreating flavivirus infection, preferably DENV, YFV, WNV, or JEVinfection and most preferably DENV, comprising administering aneffective amount of a retinoic acid analogue to a subject in needthereof, wherein the amount administered achieves a therapeuticallyeffective level in the subject, and wherein the retinoic acid analogueadministered is N-(4-hydroxyphenyl)-4-oxoretinamide.

Alternatively, the metabolites of 4-HPR may be co-administered with4-HPR and preferably work synergistically with the 4-HPR.Co-administration includes formulation of 4-HPR and one or moremetabolites as a single composition, administered as separatecompositions contemporaneously, or consecutive administration ofseparate compositions.

In an alternative embodiment, the retinoic acid analogue is a compoundaccording to the following Formula I:

wherein:R is OH, COOH, CH₂OH, CH₂CH₂OH, or CH₂COOH;carbons a-d and f-i are optionally substituted with one or more groupsselected from CH₃, OH, COOH, (CH₃)₂ and CH₂OH, or any combinationthereof, andcarbon e is optionally substituted with a C₁-C₃ alkyl group that isoptionally substituted with CH₃ and/or OH.

For the purposes of this invention, these compounds are collectivelyreferred to as 4-HPR variants.

Without wishing to be bound by any theory as to the mechanism of action,the inventors have shown that 4-HPR acts by inhibiting the associationof DENV non-structural protein 5 (NS5) protein with host cell importinproteins, and thereby blocks NS5 nuclear localisation. NS5 providesRNA-dependent RNA polymerase activity, enabling the virus to copyitself. Despite the fact that DENV replication occurs entirely withinthe cell cytoplasm, replication is highly dependent on the nuclearlocalisation of NS5 in the host cell nucleus (Pryor et al., 2007,Traffic). The inventors were the first to show that NS5 trafficking intoand out of the nucleus is dependent on interaction with specific hostcell nuclear transport receptors, and that these could thereforerepresent viable antiviral targets. In order for NS5 to enter thenucleus it must traffic through nuclear pore complexes embedded in thenuclear envelope. This is controlled by the binding of NS5 via itsnuclear localisation signals (NLS) to its specific nuclear transportproteins, the importins (IMP) α/β1 (and particularly IMPα) which in turnis critical for subsequent modulation of specific gene transcriptionrelating to modulation of the host immune response (IL-8 production andinhibition of cell cycle progression) required for efficient virusproduction. The NLS is conserved across DENV1-4 serotypes, making thisinvention advantageous in that it is effective for all circulating DENVserotypes. The NLS is also conserved across other flaviviruses such asYFV and JEV.

Accordingly, in another aspect of the invention, there is provided amethod of blocking NS5 nuclear localisation, comprising administering aneffective amount of a retinoic acid analogue to a subject, preferablythe retinoic acid analogue is 4-HPR and/or one of more metabolites of4-HPR and/or one or more 4-HPR variants according to Formula I.Typically an effective amount of a retinoic acid analogue to block NS5nuclear localisation is an amount of retinoic acid analogue thatminimises or prohibits binding of NS5 to IMPα/β1 and particularly toIMPα, thereby reducing the ability of NS5 to get in to the nucleus.

4-HPR has previously been reported to inhibit dihydroceramide desaturaseand thereby de novo ceramide synthesis. Replication of thedengue-related virus, JEV, has been shown to be dependent on ceramide;DENV replication is also tightly linked to lipid synthesis although arole for ceramide has not been examined. It is possible that inhibitionof ceramide synthesis is another mechanism of action for 4-HPRinhibition of virus replication, such as DENV replication.

Another possible mechanism of action relates to cell stress responses.4-HPR has been studied for its anti-tumour and chemo-preventativeproperties where it specifically induces apoptosis in cancerous cells.Interestingly, the inventors have shown that addition of 4-HPR toDENV-infected cells induces activation of the pro-apoptotic PERK pathwaywithin the unfolded protein response (UPR), but does not result in celldeath at concentrations that effectively remove the virus. Without beingbound by any theory, activation of the PERK pathway may facilitate hostresponses against DENV and also WNV_(KUN), with specific activation ofthe PERK arm of the UPR leading to a potent antiviral state.

In any of the treatment methods of the invention, the retinoic acidanalogue administered may consist only of 4-HPR, 4-oxo-4-HPR, or a 4-HPRvariant according to Formula I, or two or more of 4-HPR, 4-oxo-4-HPR anda 4-HPR variant according to Formula I as the only active ingredients.

Alternatively, the patient may also be receiving other treatments, suchthat the 4-HPR or compositions thereof are administered in an adjuncttherapy. Additional therapies may be those that treat the accompanyingsymptoms including, without limitation, pain relief such as aspirin,acetaminophen and codeine, and nonsteroidal anti-inflammatory drugs(NSAIDS). In this regard, there is provided a method of treatingflavivirus infection, or a method of reducing symptom severity in apatient having or suspected of having a flavivirus infection, comprisingadministering an effective amount of a retinoic acid analogue to asubject in need thereof, together with another therapeutic, wherein theamount of retinoic acid analogue administered achieves a therapeuticallyeffective level in the subject.

In a preferred embodiment, the flavivirus is one or more serotypes ofDENV, the retinoic acid analogue administered is 4-HPR and/or one ormore 4-HPR metabolites and/or 4-HPR variants according to Formula I, andthe additional therapeutic is one or more of pain relief such asaspirin, acetaminophen and codeine, and NSAIDS.

Symptoms may also be reduced in a patient infected with or suspected ofbeing infected with CHIKV by administration of a retinoic acid analogueof the invention.

In the treatment methods of the invention described herein, the methodsmay further include the step of diagnosing the subject. Diagnosis may beconducted before administration of the retinoic acid analogue, or afteradministration. Diagnosis may be made on the basis of:

-   -   symptoms    -   qRT-PCR    -   serological assays including, but not limited to ELISA and RIA.

While infection by one DENV serotype is protective of re-infection bythe same serotype, the subject may not be protected by infection withother serotypes. Subsequent infection with a different serotype isthought to result in ADE DENV infection. Accordingly, in someembodiments of the invention, the subject has had a prior DENV infectionwith a different DENV serotype. These subjects are at higher risk of ADEDENV infection. The methods of the invention are also suitable forpreventing ADE DENV infection.

ADE occurs when circulating antibodies from a previous infection with adifferent DENV serotype lead to increased viral load, caused bycross-reactive but non-neutralising antibodies. It also results in theincreased expression of genes such as those encoding the cytokinesinterleukin (IL) 8 and tumor necrosis factor (TNF) and in turn, thedevelopment of the more severe manifestations of DENV infection ofdengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS) (Medin etal., 2005, J Virol.; Wati et al., 2010, J Gen Virol.; Green and Rothman,2006, Curr Opin Infect Dis.; Clyde K et al. 2006, J Virol.). Little isknown of the mechanisms behind ADE, but recent data from the inventorsindicates a mechanistic link to DENV NS5 (unpublished observations).

Accordingly, in a further embodiment of the invention, there is provideda method for preventing ADE DENV infection, comprising administering aneffective amount of a retinoic acid analogue to a subject in needthereof. Typically the amount administered achieves a therapeuticallyeffective level in the subject to reduce the risk of ADE DENV infection.The retinoic acid analogue is preferably 4-HPR, a 4-HPR metabolite suchas 4-oxo-4-HPR, or a 4-HPR variant according to Formula I or optionally2 or more of 4-HPR, a 4-HPR metabolite and a 4-HPR variant according toFormula I.

There is also provided a use of an effective amount of a retinoic acidanalogue in the preparation of a medicament for preventing ADE DENVinfection in a subject in need thereof. The retinoic acid analogue ispreferably 4-HPR, optionally with one or more 4-HPR metabolites, or ametabolite itself, such as 4-oxo-4-HPR. The retinoic acid analogue mayalternatively be a 4-HPR variant according to Formula I on its own, orin combination with one or more of 4-HPR and a 4-HPR metabolite, such as4-oxo-4-HPR.

The inventors have shown that not only is 4-HPR able to reduce DENV whenadded to cells infected with DENV, but that treatment of cells with4-HPR prior to exposing those cells to DENV protected the cells.Accordingly, in another aspect of the invention, there is provided amethod of preventing flavivirus infection, preferably DENV, YFV, WNV, orJEV infection and most preferably DENV, comprising administering aneffective amount of a retinoic acid analogue to a subject in needthereof, wherein the amount administered achieves a prophylacticallyeffective level in the subject, thereby preventing flavivirus infection.

In an alternative embodiment, there is provided a method of preventingCHIKV infection, comprising administering an effective amount of aretinoic acid analogue to a subject in need thereof, wherein the amountadministered achieves a prophylactically effective level in the subject,thereby preventing CHIKV infection.

There is also provided a use of an effective amount of a retinoic acidanalogue in the preparation of a medicament for preventing flavivirusinfection, preferably DENV, YFV, WNV, or JEV infection and mostpreferably DENV, in a subject in need thereof. The retinoic acidanalogue is preferably 4-HPR, or one or more 4-HPR metabolites, or oneor more 4-HPR variants or 4-HPR, two or more of 4-HPR metabolites and4-HPR variants according to Formula I.

A medicament may also be prepared for preventing CHIKV infection.

The effective amount can be determined using methods known in the artand by the methods described in the examples section of thespecification.

In any of the prophylactic and therapeutic methods of the invention, theretinoic acid analogue administered may consist of 4-HPR, 4-oxo-4-HPR, a4-HPR variant of Formula I or two or more of 4-HPR, 4-oxo-4-HPR and4-HPR variants as the only active ingredients.

The retinoic acid analogues for use in the methods of the invention canbe formulated as pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents. Forconvenience, the compositions will be described in greater detail withreference to the preferred retinoic acid analogue, 4-HPR. However, ashas been detailed throughout the specification, the retinoic acidanalogue administered may also be a metabolite of 4-HPR, such as4-oxo-4-HPR. It may alternatively be a 4-HPR variant according toFormula I.

In pharmaceutical dosage forms, the 4-HPR may be administered alone or,as already mentioned above, in association or combination with otherpharmaceutically active compounds such as NSAIDS and pain relievers tohelp treat the associated symptoms of DENV, YFV, WNV, or JEV infection.Those with skill in the art will appreciate readily that effective doselevels for 4-HPR will vary as a function of the nature of the deliveryvehicle, the metabolism of the compound, the mode of delivery, thefrequency of administration and the like. As noted above, these are allfactors taken in to consideration when determining the amount to beadministered in order to achieve therapeutic or prophylactic levels.

Pharmaceutically acceptable carriers or diluents contemplated by theinvention include any diluents, carriers, excipients, and stabilizersthat are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as plasma albumin, gelatine, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

In general the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and if necessary, shaping theproduct. Formulation may be conducted by mixing at ambient temperatureat the appropriate pH, and at the desired degree of purity, withphysiologically acceptable carriers, i.e., carriers that are non-toxicto recipients at the dosages and concentrations employed.

4-HPR is preferably formulated into preparations for oral delivery withthe Lym-X-Sorb® (Lymphatic Xenobiotic AbSorbability) drug deliverysystem with controlled release (BioMolecular Products, Inc.). The 4-HPRis complexed in a 1:1 molar ratio with the Lym-X-Sorb, forming stableparticles 10 to 70 nm in size, which are readily absorbed in thepresence of sodium bicarbonate and bile salt. The 4-HPR complex istherefore better protected from digestive degradation(biomolecularproducts.com website).

In the treatment methods of the invention the 4-HPR compositions areadministered to an infected human subject in need of treatment for acertain period of time. That period of time can vary on the type ofviral infection, the severity of the viral infection, the stage ofinfection, and the responsiveness of the subject to treatment. Ingeneral, the subject will be treated for a period of 5 to 14 days. Uponcompleting treatment, the subject can optionally be tested for thepresence of virus to confirm the success of treatment.

In the case of treating a subject to prevent them from becoming infectedwith, for example, DENV, the subject may take a daily dose of 4-HPR foras long as they are at risk of becoming infected with DENV. For example,for subjects going to locations endemic for DENV, the subject will takethe 4-HPR for the entire period they are in the location. Preferably,administration will begin 1 to 5 days prior to arriving in the location,and will continue for 1 to 14 days after leaving the location.

The dose to be administered, for either treatment or prophylaxis, may bein the form of single doses of higher concentration, or divided doses oflower concentration. The concentration of any given dose will depend onthe frequency of administration.

In another embodiment there is provided a kit or article of manufactureincluding a retinoic acid analogue, preferably 4-HPR, or pharmaceuticalcomposition as described above.

In other embodiments there is provided a kit for use in a therapeuticapplication mentioned above, the kit including:

-   -   a container holding the retinoic acid analogue, preferably        4-HPR, or pharmaceutical composition thereof; and    -   a label or package insert with instructions for use in the        methods of the invention described herein.

In certain embodiments the kit may contain one or more further activeprinciples or ingredients for treatment of DENV, YFV, WNV, or JEVinfections as described above.

The kit or “article of manufacture” may comprise a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, blister pack,etc. The containers may be formed from a variety of materials such asglass or plastic. The container holds a retinoic acid analogue,preferably 4-HPR, or pharmaceutical composition thereof which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The labelor package insert indicates that the retinoic acid analogue, preferably4-HPR, or pharmaceutical composition thereof is used for treating thecondition of choice. In one embodiment, the label or package insertincludes instructions for use and indicates that the retinoic acidanalogue, preferably 4-HPR, or pharmaceutical composition thereof can beused to treat or prevent the methods described above.

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings.

EXAMPLES Example 1 Effect of Retinoic Acid Analogues on NS5-IMPα/β1Association

To investigate the structure/activity relationship for 4-HPR and4-oxo-4-HPR, all-trans retinoic acid (ATRA; the parent compound of4-HPR) and N-(4-methoxyphenyl) retinamide (4-MPR; a closely relatedanalogue of 4-HPR) were compared in their ability to inhibit associationof NS5 with IMPα/β1:

ALPHASCREEN®, a luminescent bead-based assay that measures directprotein-protein interactions, was used to examine the binding of 3 nMHis₆-NS5 to 10 nM IMPα/β1 (IMPα/β1 heterodimer containing biotinylatedIMPβ1) in the presence of increasing concentrations of 4-HPR, 4-MPR orATRA (FIG. 1A), or 4-oxo-4-HPR, 4-HPR or 4-MPR (FIG. 1B). Data are themean+standard deviation (SD) of triplicate wells from a single typicalexperiment.

4-HPR and 4-oxo-4-HPR were the only retinoic acid analogues able toblock association of DENV-2 NS5 with IMPα/β1 (FIG. 1A and FIG. 1B). Incontrast to 4-HPR and 4-oxo-4-HPR, neither ATRA nor 4-MPR were able todisrupt association of these proteins, underlining the specificity ofthe interaction.

Example 2 Specific Inhibition of DENV-2 NS5 Association with IMPα

To investigate the specificity of 4-HPR inhibition of NS5-importinassociation, the binding of 30 nM His₆-NS5 to 10 nM IMPα/β1, (IMPα/β1heterodimer containing biotinylated IMPβ1), 3 nM IMPαΔIBB (biotinylatedIMPα with the IMPβ-binding domain removed), or 3 nM biotinylated IMPβ1,in the presence of increasing concentrations of 4-HPR, was investigated.4-HPR was found to interfere with association of NS5 and IMPα/β1, aswell as NS5 and IMPαΔIBB (a truncated form of IMPα which lacks theauto-inhibitory IMPβ-binding domain), with 50% inhibition of bindingoccurring at similar concentrations (average IC₅₀ of 1.2 and 1.6 μM,respectively, from 3 independent experiments; representative assayshown), while NS5 binding to IMPβ1 alone was largely unaffected by thepresence of 4-HPR.

These further investigations determined that 4-HPR prevents NS5 frombinding to the NLS binding site on IMPα in vitro, by targeting either orboth NS5 and IMPα (FIG. 2).

Example 3 4-HPR Antiviral Specificity

To test the specificity of 4-HPR and 4-oxo-4-HPR for anti-DENV activityVero cells were infected with DENV-2 with a multiplicity of infection(MOI) of 1, in the presence of drug diluent (untreated) or the indicatedcompounds (10 μM in FIG. 3A, or 10 μM or 50 μM as indicated in FIG. 3B)for 2 h, followed by removal of the virus/drug inoculums, and additionof fresh 2% FCS maintenance medium. At 24 h post infection (p.i.) theculture medium was collected and the viral RNA extracted using QIAampviral RNA mini kit (Qiagen). Absolute RNA copies present in each samplewere determined by Taqman One Step RT-PCR (Applied Biosystems), as anestimate of virus titre by extrapolation from a standard curve generatedfrom in vitro transcribed DENV RNA. Data shown are the mean+standarderror of the mean (SEM) for 3 (FIG. 3A) or 2 (FIG. 3B) independentexperiments. Statistical analysis (Student's t test) was performed usingGraphPad Prism software. 4-HPR was found to have potent antiviralactivity in this context, with 10 μM of the compound causing a 2-logreduction in viral RNA copies at 24 h p.i. (****p<0.0001) (FIG. 3A).4-oxo-4-HPR was also found to have significant antiviral activity, with5 μM treatment causing a 1-log reduction in viral RNA copies at 24 h p.i(**p<0.01), similar to 5 μM 4-HPR (FIG. 3B). By contrast, addition of 10μM ATRA, 4-MPR, or 2 other 4-HPR analogues, arotinoid acid (AA) and13-cis retinoic acid (13-cRA), had no effect on DENV infection (FIG. 3A;NS, not significant).

Example 4 Toxicity Analysis

To determine the effect of 4-HPR on cell viability, and to ensure thatthe reduced viral titres were not due to compound toxicity, XTT reagentwas used to measure mitochondrial activity of drug treated cells.Briefly, Vero cells were incubated with 0, 1, 10 or 25 μM 4-HPR for 24h. XTT reagent was added directly to cells and incubated for 6 h at 37°C. prior to spectrophotometric analysis. Data shown are the mean+SD of asingle experiment performed in triplicate wells.

Treatment of Vero cells for 24 h with up to 25 μM 4-HPR had no effect oncell viability (FIG. 4). This is indicative of a highly favourabletoxicity profile for 4-HPR and confirms the previous favourable toxicityprofiles seen where 4-HPR has been used to treat various cancers,including in children, at concentrations of up to 4,000 mg/m²/day,administered daily for 28 days (Formelli et al., 2008, Clin, CancerChemother Pharmacol).

Example 5 4-HPR as a Prophylactic and Treatment for Dengue VirusInfections

To investigate whether 4-HPR can inhibit DENV-2 replication when addedprior to infection or post infection (p.i.), 10 μM drug or DMSO (drugdiluent) was added to Vero cells 2 h prior to infection, for 2 h duringinfection, 2 h p.i., or 24 h p.i. In each instance the drug wasincubated with the cells for 2 h then removed. Infections were performedfor 2 h (MOI of 1) followed by analysis of intracellular viral RNA byqRT-PCR 48 h p.i. Data shown are the mean+SD from one experiment,representative of three independent assays.

Treatment of cells with 4-HPR prior to, during, or up to 24 h p.i causeda substantial reduction of intracellular DENV-2 RNA copies (FIG. 5A).This suggests that 4-HPR can have prophylactic activity to protectagainst infection, as well as being effective when added p.i. as aninfection treatment.

To gain further insight into the mode of action of 4-HPR, the humanliver carcinoma cell line, Huh-7 cells were infected with DENV-2 (MOI of1), followed by delayed addition of drug diluent DMSO (DENV only), 7.5μM 4-HPR, 7.5 μM 4-MPR or 10 μM NITD008 (a previously describedadenosine nucleoside inhibitor), 12 h post infection. Cells were lysedat the indicated times, RNA was extracted by Trizol and analysed by qPCRwith fold change in expression of NS 1 calculated relative to theuntreated control (FIG. 5B). Concurrently, culture medium was collectedand viral titres determined by plaque assay (FIG. 5C).

In the untreated DENV control, or infected cells treated with 7.5 μM4-MPR, viral RNA levels increased steadily until 60 h p.i. (FIG. 5B). Incontrast, addition of 7.5 μM 4-HPR to Huh-7 cells at 12 h p.i. resultedin significantly reduced viral RNA levels relative to the infectioncontrol and 4-MPR-treated cells, with a 1-log reduction observed within12 h (24 h p.i., *p<0.05). Interestingly, the amount of viral RNApresent did not increase between 24 h and 60 h p.i. This result wascomparable to the reduced RNA levels obtained in infected cells treatedwith the polymerase inhibitor NITD008 (***p<0.005).

Parallel analysis of the amount of infectious virus produced revealedresults consistent with reduced RNA levels, where delayed addition of4-HPR resulted in significantly reduced virus titres over the 60 hinfection period, relative to the untreated and 4-MPR controls(***p<0.001, FIG. 5C). A maximal reduction was reached at 60 h p.i.where the virus titre was 3-logs lower in 4-HPR-treated cells comparedto the untreated and 4-MPR-treated controls, showing similar reductionsto the NITD008 control. This finding has implications for the potentialtherapeutic use of this compound, suggesting that the compound may beefficacious in treating an established infection.

Example 6 4-HPR Blocks Replication of all Four Circulating DENVSerotypes

There are 4 circulating serotypes of DENV, and infection with oneserotype is not protective of infection with another. It is thereforeimportant that any prophylactic or treatment is effective against eachserotype.

To investigate this, BHK-21 cells were infected with DENV serotypes 1,2, 3 or 4 (MOI of 0.3) for 1 h in the presence of 4-HPR or 4-MPR(concentrations as indicated in FIG. 6A). Virus inoculum was removed andfresh media containing the indicated compounds was added. Virusinfection rates were determined 48 h p.i. using a cell-based flavivirusimmunodetection assay. Briefly, cells were fixed and stained for DENVstructural protein, E, followed by addition of Alexa Fluor 488anti-mouse IgG. Viral antigen quantification was performed usingImagexpress. Data shown are representative of a single experiment.

4-HPR was found to block replication of all 4 DENV serotypes in BHK-21cells with a low EC₅₀. DENV-4 was found to be inhibited with the lowestEC₅₀ (0.84 μM) while DENV-2 was inhibited with the highest EC₅₀ (2.24μM). Consistent with the previous findings for DENV-2, it was found that4-MPR did not have any effect on the replication of DENV serotypes 1, 2,3, or 4. These findings demonstrate that 4-HPR is similarly effectiveagainst all circulating DENV serotypes.

To ensure the antiviral activity of 4-HPR is not cell-line specificHuh-7 cells were also infected with DENV-1, 2, 3 or 4 (MOI of 0.3) inthe presence of increasing concentrations of 4-HPR, with infectiousvirus titres determined by plaque assay at 48 h p.i. All 4 serotypes ofDENV were restricted with similar EC₅₀ values (average of 2.6, 2.1, 1.4and 2.1 μM for DENV 1, 2, 3 and 4, respectively, from 2 independentexperiments; see FIG. 6B, representative data shown), consistent withthe findings in BHK-21 cells.

Example 7 4-HPR is Effective Against DENV-1/Antibody-Dependent EnhancedInfection

In a cell culture model of antibody-dependent enhanced (ADE) DENVinfection, THP-1 human monocyte cells infected with DENV-1 in thepresence of sub-neutralising levels of a humanized version of the 4G2antibody, which is cross-reactive with flavivirus envelope proteins(Paradkar et al., 2010 Bio Sci Rep.), show increased virus productioncompared to cells infected with virus alone. This model mimics the ADEphenomenon thought to occur in patients infected with a second serotypeof DENV, which is associated with more severe DENV disease.

Briefly, humanized 4G2 antibody (0.05 μg/ml) was combined with DENV-1(MOI of 10), and 4-HPR (concentrations as indicated in FIG. 7) inserum-free media, and incubated on ice for 1 h for the immune complex toform. Immune complex inoculums were added to THP-1 cells and incubatedfor 48 h. Supernatants were collected after 2 days and infectious virusproduction was quantified by plaque assay.

4-HPR was highly effective in blocking ADE-mediated DENV-1 infection inTHP-1 cells (FIG. 7). A low EC₅₀ of 0.85 μM was determined. This findingindicates that 4-HPR is effective in restricting DENV replication, evenwhen infection occurs via ADE, which is considered to be linked to moresevere disease outcomes.

Example 8 4-HPR Provides Protection Against DENV Infection Ex Vivo andIn Vivo

The antiviral activity of 4-HPR was investigated in the morephysiologically relevant human system of peripheral blood mononuclearcells (PBMCs), a major target of DENV infection in humans.

PBMCs were prepared from a healthy donor buffy coat using a Ficollgradient, prior to ADE-mediated DENV-1 infection (MOI of 10) in thepresence of increasing concentrations of 4-HPR or 4-MPR, and infectiousvirus titres determined by plaque assay 48 h later. In this system 4-HPRwas determined to be highly effective with an ex vivo EC₅₀ value of 0.81μM (FIG. 8A, viral titre is plotted on the left hand y-axis, blackline), in contrast to no reduction in infected PBMCs treated with 4-MPR.Cytotoxicity analysis of 4-HPR in this system indicated a CC₅₀ valueof >13 μM (FIG. 8A, cell survival is plotted on the right hand y-axis,grey line), underlining the fact that the loss of infectious virus isnot due to induction of cell death.

Mice have previously been used as an in vivo model for 4-HPRpharmacokinetics. To examine the effect of 4-HPR on DENV infection invivo, a lethal mouse infection model, which uses a mouse adapted DENV-2strain (S221), to infect Sv/129 mice deficient in type I and IIinterferon receptors (AG129) was used (Zellweger et al., 2010, Cell HostMicrobe). Viral infection in this system is enhanced by intraperitonealdelivery of a sub-neutralizing concentration of 4G2 antibody 1 day priorto infection, resulting in peak viremia 3 days p.i. and death ofinfected mice by day 5. Here, mice were treated with 20 mg/kg of 4-HPR,administered orally at the time of infection, then once (QD) or twice(BID) daily for 5 consecutive days. Consistent with previous reports,infected, untreated mice all died by day 5 (FIG. 8B). Once dailytreatment of mice with 4-HPR was sufficient to provide protection to 20%of mice, which recovered from the infectious challenge, whereasincreasing the dose to 2 treatments daily, improved protection to 70% (7of 10 mice recovered from the infectious challenge).

Analysis of plasma viral titres in mice 3 days p.i. revealed a smallreduction in the average viral titre in the QD treatment group, whilethe BID treatment group was found to have an average viral titre 65%lower than that of the untreated group (**p=0.002, FIG. 8C), consistentwith 4-HPR providing protection by lowering the amount of infectiousDENV present in the infected and treated mice. Data are the mean+SEM.Statistical analysis (Student's t test) was performed using GraphPadPrism software (ns, not significant).

Example 9 4-HPR is Effective Against the Arboviruses Chikungunya Virus(CHIKV) and West Nile Virus (WNV)

HEK293T cells were treated with 5 μM 4-HPR or 4-MPR for 2 h, theninfected with CHIKV for 1 h (MOI of 0.1) in the absence of compound(FIG. 9A). Alternatively, HEK293T cells were infected with CHIKV for 1 h(MOI of 0.1) without pre-treating the cells with compound (FIG. 9B). Inboth instances, virus inoculums were removed, then fresh mediumcontaining the indicated compound was added (FIG. 9A and FIG. 9B).Infectious virus titres were determined by plaque assay 24 h p.i. Dataare the mean+standard deviation of one experiment performed in duplicatewells, represented as plaque forming units per ml (PFU/ml).

4-HPR (5 μM) was highly effective in restricting the production ofinfectious CHIKV, whether it was added to cells prior to infection orafter infection. Compared to untreated controls, 4-HPR reduced theamount of infectious CHIKV produced by almost 100% in the cells thatwere pre-treated with compound, and by almost 95% in cells to which4-HPR was added after infection. 4-MPR did not reduce CHIKV titres whenadded either prior to or after infection. Given that 4-HPR was effectivein reducing CHIKV titres when added after virus attachment to cells,(FIG. 9B) it is likely that 4-HPR is effective at the level of RNAreplication, similar to the mode of action of 4-HPR against DENV. 4-HPRalso showed activity against the DENV-related flavivirus, West Nilevirus, Kunjin strain (WNV_(KUN); FIG. 9C), demonstrating an 89%reduction in infectious virus produced following a 4 h incubation ofVero cells with 10 μM 4-HPR at the time of infection. Again, 4-MPR wasfound to have no antiviral activity.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

REFERENCES

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The invention claimed is:
 1. A method of preventing dengue virus (DENV)in a subject at risk of being exposed to DENV, comprising: administeringa daily dose of N-(4-hydroxyphenyl) retinamide (4-HPR) to the subject ofup to 4,000 mg/m²/day during the period of time in which the subject isat risk of exposure, wherein the subject is not being administered ananti-viral agent other than 4-HPR.
 2. The method of claim 1, furthercomprising administering the daily dose of 4-HPR of up to 4,000mg/m²/day for 1 to 5 days prior to being exposed to DENV.
 3. The methodof claim 1, further comprising administering the daily dose of 4-HPR ofup to 4,000 mg/m²/day for 1 to 14 days after being exposed to DENV. 4.The method of claim 1, wherein the subject has had a prior DENVinfection.
 5. The method of claim 1, wherein the dose administered is asingle daily dose or divided daily doses.
 6. A method of preventingdengue virus (DENV) in a subject at risk of being exposed to DENV,comprising: administering a daily dose of 4-HPR of up to 4,000 mg/m²/dayfor 1 to 5 days prior to being exposed to DENV; administering a dailydose of N-(4-hydroxyphenyl) retinamide (4-HPR) to the subject of up to4,000 mg/m²/day during the period of time in which the subject is atrisk of exposure; and administering a daily dose of 4-HPR of up to 4,000mg/m²/day for 1 to 14 days after being exposed to DENV wherein thesubject is not being administered an anti-viral agent other than 4-HPR.7. The method of claim 6, wherein the subject has had a prior DENVinfection.
 8. The method of claim 6, wherein the dose administered is asingle daily dose or divided daily doses.